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1.
Org Biomol Chem ; 19(40): 8821-8829, 2021 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-34585207

RESUMO

Native chemical ligation (NCL) enables the total chemical synthesis of proteins. However, poor peptide segment solubility remains a frequently encountered challenge. Here we introduce a traceless linker that can be temporarily attached to Glu side chains to overcome this problem. This strategy employs a new tool, Fmoc-Glu(AlHx)-OH, which can be directly installed using standard Fmoc-based solid-phase peptide synthesis. The incorporated residue, Glu(AlHx), is stable to a wide range of chemical protein synthesis conditions and is removed through palladium-catalyzed transfer under aqueous conditions. General handling characteristics, such as efficient incorporation, stability and rapid removal were demonstrated through a model peptide modified with Glu(AlHx) and a Lys6 solubilizing tag. Glu(AlHx) was incorporated into a highly insoluble peptide segment during the total synthesis of the bacteriocin AS-48. This challenging peptide was successfully synthesized and folded, and it has comparable antimicrobial activity to the native AS-48. We anticipate widespread use of this easy-to-use, robust linker for the preparation of challenging synthetic peptides and proteins.


Assuntos
Ácido Glutâmico
2.
Org Biomol Chem ; 17(48): 10237-10244, 2019 12 28.
Artigo em Inglês | MEDLINE | ID: mdl-31793605

RESUMO

The application of solid-phase peptide synthesis and native chemical ligation in chemical protein synthesis (CPS) has enabled access to synthetic proteins that cannot be produced recombinantly, such as site-specific post-translationally modified or mirror-image proteins (D-proteins). However, CPS is commonly hampered by aggregation and insolubility of peptide segments and assembly intermediates. Installation of a solubilizing tag consisting of basic Lys or Arg amino acids can overcome these issues. Through the introduction of a traceless cleavable linker, the solubilizing tag can be selectively removed to generate native peptide. Here we describe the synthesis of a next-generation amine-reactive linker N-Fmoc-2-(7-amino-1-hydroxyheptylidene)-5,5-dimethylcyclohexane-1,3-dione (Fmoc-Ddap-OH) that can be used to selectively introduce semi-permanent solubilizing tags ("helping hands") onto Lys side chains of difficult peptides. This linker has improved stability compared to its predecessor, a property that can increase yields for multi-step syntheses with longer handling times. We also introduce a new linker cleavage protocol using hydroxylamine that greatly accelerates removal of the linker. The utility of this linker in CPS was demonstrated by the preparation of the synthetically challenging Shiga toxin subunit B (StxB) protein. This robust and easy-to-use linker is a valuable addition to the CPS toolbox for the production of challenging synthetic proteins.


Assuntos
Peptídeos/química , Subunidades Proteicas/síntese química , Toxina Shiga/síntese química , Técnicas de Síntese em Fase Sólida/métodos , Sequência de Aminoácidos , Arginina/química , Cicloexanonas/química , Hidroxilamina/química , Lisina/química , Solubilidade
3.
Scand J Caring Sci ; 33(1): 57-66, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30320477

RESUMO

BACKGROUND: Sexual dysfunction is common in patients diagnosed with cancer or chronic disease, having psychological, emotional and social ramifications. The consequences are ultimately significant impaired quality of life and reduced mental well-being, and it therefore requires professional attention. However, evidence suggests that sexuality is a taboo subject in the healthcare system, as healthcare professionals and patients rarely initiate conversations on this subject. AIM: Therefore, the purpose of this study was to understand how patients diagnosed with cancer or chronic disease experiencing sexual dysfunction experience sexuality as a taboo subject in the healthcare system. METHODS: Ten women diagnosed with cancer or chronic disease experiencing sexual dysfunction were recruited from the Sexological Centre in Aalborg, Denmark. Using an interview guide, semi-structured interviews were conducted and a qualitative thematic analysis was performed. RESULTS: The analysis revealed that sexuality is a sensitive and taboo subject in the Danish healthcare system. Some patients initiated conversations about their sexuality, but felt rejected by healthcare professionals, who dismissed these initiatives, whereas other patients were reluctant to discuss sexuality, but did not experience healthcare professionals bringing up sexuality either. Despite these differences between patients' experiences, sexuality constituted a taboo subject, as patients or healthcare professionals deliberately avoided discussing the patients' sexuality-related issues. CONCLUSIONS: Communication about sexuality is essential for improving patients' well-being, and healthcare professionals should routinely address sexuality and sexual intimacy with patients diagnosed with cancer or chronic disease in order to break the taboo and accommodate patients' needs.


Assuntos
Doença Crônica/psicologia , Comunicação , Neoplasias/complicações , Neoplasias/psicologia , Disfunções Sexuais Fisiológicas/psicologia , Sexualidade/psicologia , Tabu/psicologia , Adulto , Idoso , Dinamarca , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pesquisa Qualitativa , Adulto Jovem
4.
J Electrocardiol ; 51(6): 1053-1054, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30497729

RESUMO

Leadless pacemaker implantation represents an important advancement in the treatment of bradycardia, and occupy an increasing part in the clinic. Major adverse effects associated with leadless pacemaker implantation are rare, with a serious complication being pericardial effusion. We present a case of a patient who had a leadless pacemaker implanted, which induced ventricular tachycardia and cardiac arrest during hospitalization.


Assuntos
Parada Cardíaca/etiologia , Marca-Passo Artificial/efeitos adversos , Taquicardia Ventricular/etiologia , Idoso de 80 Anos ou mais , Bradicardia/terapia , Eletrocardiografia , Desenho de Equipamento , Falha de Equipamento , Humanos , Masculino , Taquicardia Ventricular/diagnóstico
5.
Bioorg Med Chem ; 25(18): 4946-4952, 2017 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-28651912

RESUMO

The scope of chemical protein synthesis (CPS) continues to expand, driven primarily by advances in chemical ligation tools (e.g., reversible solubilizing groups and novel ligation chemistries). However, the design of an optimal synthesis route can be an arduous and fickle task due to the large number of theoretically possible, and in many cases problematic, synthetic strategies. In this perspective, we highlight recent CPS tool advances and then introduce a new and easy-to-use program, Aligator (Automated Ligator), for analyzing and designing the most efficient strategies for constructing large targets using CPS. As a model set, we selected the E. coli ribosomal proteins and associated factors for computational analysis. Aligator systematically scores and ranks all feasible synthetic strategies for a particular CPS target. The Aligator script methodically evaluates potential peptide segments for a target using a scoring function that includes solubility, ligation site quality, segment lengths, and number of ligations to provide a ranked list of potential synthetic strategies. We demonstrate the utility of Aligator by analyzing three recent CPS projects from our lab: TNFα (157 aa), GroES (97 aa), and DapA (312 aa). As the limits of CPS are extended, we expect that computational tools will play an increasingly important role in the efficient execution of ambitious CPS projects such as production of a mirror-image ribosome.


Assuntos
Biologia Computacional/métodos , Proteínas/síntese química , Software , Chaperonina 10/síntese química , Chaperonina 10/química , Chaperonina 60/síntese química , Chaperonina 60/química , Escherichia coli/metabolismo , Proteínas/química , Proteínas Ribossômicas/síntese química , Proteínas Ribossômicas/química , Fator de Necrose Tumoral alfa/síntese química , Fator de Necrose Tumoral alfa/química
6.
Proc Natl Acad Sci U S A ; 111(32): 11679-84, 2014 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-25071217

RESUMO

Mirror-image proteins (composed of D-amino acids) are promising therapeutic agents and drug discovery tools, but as synthesis of larger D-proteins becomes feasible, a major anticipated challenge is the folding of these proteins into their active conformations. In vivo, many large and/or complex proteins require chaperones like GroEL/ES to prevent misfolding and produce functional protein. The ability of chaperones to fold D-proteins is unknown. Here we examine the ability of GroEL/ES to fold a synthetic d-protein. We report the total chemical synthesis of a 312-residue GroEL/ES-dependent protein, DapA, in both L- and D-chiralities, the longest fully synthetic proteins yet reported. Impressively, GroEL/ES folds both L- and D-DapA. This work extends the limits of chemical protein synthesis, reveals ambidextrous GroEL/ES folding activity, and provides a valuable tool to fold d-proteins for drug development and mirror-image synthetic biology applications.


Assuntos
Enzimas/biossíntese , Enzimas/química , Chaperonas Moleculares/metabolismo , Dobramento de Proteína , Sequência de Aminoácidos , Aminoácidos/química , Fenômenos Biofísicos , Chaperonina 10/metabolismo , Chaperonina 60/metabolismo , Enzimas/genética , Proteínas de Escherichia coli/biossíntese , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Hidroliases/biossíntese , Hidroliases/química , Hidroliases/genética , Modelos Moleculares , Dados de Sequência Molecular , Ácidos Picolínicos/metabolismo , Estrutura Quaternária de Proteína , Estereoisomerismo
7.
J Am Chem Soc ; 138(36): 11775-82, 2016 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-27532670

RESUMO

Although native chemical ligation (NCL) and related chemoselective ligation approaches provide an elegant method to stitch together unprotected peptides, the handling and purification of insoluble and aggregation-prone peptides and assembly intermediates create a bottleneck to routinely preparing large proteins by completely synthetic means. In this work, we introduce a new general tool, Fmoc-Ddae-OH, N-Fmoc-1-(4,4-dimethyl-2,6-dioxocyclo-hexylidene)-3-[2-(2-aminoethoxy)ethoxy]-propan-1-ol, a heterobifunctional traceless linker for temporarily attaching highly solubilizing peptide sequences ("helping hands") onto insoluble peptides. This tool is implemented in three simple and nearly quantitative steps: (i) on-resin incorporation of the linker at a Lys residue ε-amine, (ii) Fmoc-SPPS elongation of a desired solubilizing sequence, and (iii) in-solution removal of the solubilizing sequence using mild aqueous hydrazine to cleave the Ddae linker after NCL-based assembly. Successful introduction and removal of a Lys6 helping hand is first demonstrated in two model systems (Ebola virus C20 peptide and the 70-residue ribosomal protein L31). It is then applied to the challenging chemical synthesis of the 97-residue co-chaperonin GroES, which contains a highly insoluble C-terminal segment that is rescued by a helping hand. Importantly, the Ddae linker can be cleaved in one pot following NCL or desulfurization. The purity, structure, and chaperone activity of synthetic l-GroES were validated with respect to a recombinant control. Additionally, the helping hand enabled synthesis of d-GroES, which was inactive in a heterochiral mixture with recombinant GroEL, providing additional insight into chaperone specificity. Ultimately, this simple, robust, and easy-to-use tool is expected to be broadly applicable for the synthesis of challenging peptides and proteins.


Assuntos
Proteínas/química , Proteínas/síntese química , Sequência de Aminoácidos , Técnicas de Química Sintética , Fluorenos/química , Dobramento de Proteína , Proteínas Ribossômicas/síntese química , Proteínas Ribossômicas/química , Solubilidade , Proteínas Virais/química
8.
Org Biomol Chem ; 14(23): 5298-303, 2016 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-27211891

RESUMO

Tumor Necrosis Factor alpha (TNFα) is an inflammatory cytokine that plays a central role in the pathogenesis of chronic inflammatory disease. Here we describe the chemical synthesis of l-TNFα along with the mirror-image d-protein for use as a phage display target. The synthetic strategy utilized native chemical ligation and desulfurization to unite three peptide segments, followed by oxidative folding to assemble the 52 kDa homotrimeric protein. This synthesis represents the foundational step for discovering an inhibitory d-peptide with the potential to improve current anti-TNFα therapeutic strategies.


Assuntos
Biblioteca de Peptídeos , Fator de Necrose Tumoral alfa/química , Fator de Necrose Tumoral alfa/síntese química , Modelos Moleculares , Estrutura Secundária de Proteína , Estereoisomerismo
9.
Proc Natl Acad Sci U S A ; 108(49): E1339-48, 2011 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-22042871

RESUMO

The adult hippocampus plays a central role in memory formation, synaptic plasticity, and neurogenesis. The subgranular zone of the dentate gyrus contains neural progenitor cells with self-renewal and multilineage potency. Transgene expression of familial Alzheimer's disease-linked mutants of ß-amyloid precursor protein (APP) and presenilin-1 leads to a significant inhibition of neurogenesis, which is potentially linked to age-dependent memory loss. To investigate the effect of neurogenesis on cognitive function in a relevant disease model, FGF2 gene is delivered bilaterally to the hippocampi of APP+presenilin-1 bigenic mice via an adenoassociated virus serotype 2/1 hybrid (AAV2/1-FGF2). Animals injected with AAV2/1-FGF2 at a pre- or postsymptomatic stage show significantly improved spatial learning in the radial arm water maze test. A neuropathological investigation demonstrates that AAV2/1-FGF2 injection enhances the number of doublecortin, BrdU/NeuN, and c-fos-positive cells in the dentate gyrus, and the clearance of fibrillar amyloid-ß peptide (Aß) in the hippocampus. AAV2/1-FGF2 injection also enhances long-term potentiation in another APP mouse model (J20) compared with control AAV2/1-GFP-injected littermates. An in vitro study confirmed the enhanced neurogenesis of mouse neural stem cells by direct AAV2/1-FGF2 infection in an Aß oligomer-sensitive manner. Further, FGF2 enhances Aß phagocytosis in primary cultured microglia, and reduces Aß production from primary cultured neurons after AAV2/1-FGF2 infection. Thus, our data indicate that virus-mediated FGF2 gene delivery has potential as an alternative therapy of Alzheimer's disease and possibly other neurocognitive disorders.


Assuntos
Doença de Alzheimer/metabolismo , Transtornos Cognitivos/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Hipocampo/metabolismo , Doenças Neurodegenerativas/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/terapia , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Células Cultivadas , Transtornos Cognitivos/genética , Transtornos Cognitivos/terapia , Giro Denteado/metabolismo , Giro Denteado/patologia , Dependovirus/genética , Modelos Animais de Doenças , Fator 2 de Crescimento de Fibroblastos/genética , Terapia Genética/métodos , Vetores Genéticos/genética , Hipocampo/patologia , Hipocampo/fisiopatologia , Humanos , Imuno-Histoquímica , Potenciação de Longa Duração/fisiologia , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos da Linhagem 129 , Camundongos Transgênicos , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/terapia , Neurogênese/fisiologia , Neurônios/metabolismo , Neurônios/patologia , Presenilina-1/genética , Presenilina-1/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo
10.
Proc Natl Acad Sci U S A ; 108(7): 2729-34, 2011 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-21282621

RESUMO

Hydrogen peroxide is thought to regulate cellular processes by direct oxidation of numerous cellular proteins, whereas antioxidants, most notably thiol peroxidases, are thought to reduce peroxides and inhibit H(2)O(2) response. However, thiol peroxidases have also been implicated in activation of transcription factors and signaling. It remains unclear if these enzymes stimulate or inhibit redox regulation and whether this regulation is widespread or limited to a few cellular components. Herein, we found that Saccharomyces cerevisiae cells lacking all eight thiol peroxidases were viable and withstood redox stresses. They transcriptionally responded to various redox treatments, but were unable to activate and repress gene expression in response to H(2)O(2). Further studies involving redox transcription factors suggested that thiol peroxidases are major regulators of global gene expression in response to H(2)O(2). The data suggest that thiol peroxidases sense and transfer oxidative signals to the signaling proteins and regulate transcription, whereas a direct interaction between H(2)O(2) and other cellular proteins plays a secondary role.


Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Peróxido de Hidrogênio/toxicidade , Peroxidases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Transdução de Sinais/efeitos dos fármacos , Sequência de Bases , Modelos Biológicos , Dados de Sequência Molecular , Mutagênese , Análise de Sequência com Séries de Oligonucleotídeos , Estresse Oxidativo/genética , Peroxidases/deficiência , Fenótipo , Proteínas Ribossômicas/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Análise de Sequência de DNA , Transdução de Sinais/fisiologia
11.
Chem Sci ; 15(9): 3214-3222, 2024 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-38425513

RESUMO

We developed a new cysteine-specific solubilizing tag strategy via a cysteine-conjugated succinimide. This solubilizing tag remains stable under common native chemical ligation conditions and can be efficiently removed with palladium-based catalysts. Utilizing this approach, we synthesized two proteins containing notably difficult peptide segments: interleukin-2 (IL-2) and insulin. This IL-2 chemical synthesis represents the simplest and most efficient approach to date, which is enabled by the cysteine-specific solubilizing tag to synthesize and ligate long peptide segments. Additionally, we synthesized a T8P insulin variant, previously identified in an infant with neonatal diabetes. We show that T8P insulin exhibits reduced bioactivity (a 30-fold decrease compared to standard insulin), potentially contributing to the onset of diabetes in these patients. In summary, our work provides an efficient tool to synthesize challenging proteins and opens new avenues for exploring research directions in understanding their biological functions.

12.
Curr Opin Psychol ; 50: 101556, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36774853

RESUMO

Nostalgia is not a conventional topic of sociological analysis, and sociologists writing on nostalgia often rely on insights on the topic generated in neighboring disciplines. However, there is in fact relevant sociological work on nostalgia conducted by sociologists, and this article reviews is. Illustative examples of sociological topics relating to nostalgia are as follows: nostalgia and the past, examining how historical recollection or reconstruction and memory shape nostalgia, nostalgia and experiences of personal and cultural loss; nostalgia and individual or collective identity; nostalgia and social change; and nostalgia and politics.


Assuntos
Sociologia , Humanos , Mudança Social
13.
Methods Mol Biol ; 2530: 81-107, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35761044

RESUMO

Maintaining high, or even sufficient, solubility of every peptide segment in chemical protein synthesis (CPS) remains a critical challenge; insolubility of just a single peptide segment can thwart a total synthesis venture. Multiple approaches have been used to address this challenge, most commonly by employing a chemical tool to temporarily improve peptide solubility. In this chapter, we discuss chemical tools for introducing semipermanent solubilizing sequences (termed helping hands) at the side chains of Lys and Glu residues. We describe the synthesis, incorporation by Fmoc-SPPS, and cleavage conditions for utilizing these two tools. For Lys sites, we discuss the Fmoc-Ddap-OH dimedone-based linker, which is achiral, synthesized in one step, can be introduced directly at primary amines, and is removed using hydroxylamine (or hydrazine). For Glu sites, we detail the new Fmoc-SPPS building block, Fmoc-Glu(AlHx)-OH, which can be prepared in an efficient process over two purifications. Solubilizing sequences are introduced directly on-resin and later cleaved with palladium-catalyzed transfer under aqueous conditions to restore a native Glu side chain. These two chemical tools are straightforward to prepare and implement, and we anticipate continued usage in "difficult" peptide segments following the protocols described herein.


Assuntos
Peptídeos , Proteínas , Peptídeos/química , Solubilidade
14.
FASEB J ; 24(8): 3093-102, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20371618

RESUMO

Cytokines play an emerging role as neurotransmitters, neuromodulators, and neurohormones in the brain. This paradigm shift in cytokine function offers a new framework to understand their roles in ameliorating neurodegenerative disorders, such as Alzheimer's disease (AD). Molecular adjuvant therapy of AD animal models with glatiramer acetate induces anti-inflammatory responses and therapeutic effects. Although these effects are potentially mediated through anti-inflammatory cytokine signaling, the exact molecular identities and pathways are poorly understood. Here, we show that virus-mediated expression of the mouse interleukin (IL)-4 gene in beta-amyloid precursor protein + presenilin-1 (APP+PS1) bigenic mice attenuates AD pathogenesis. Introduction of an adeno-associated viral (AAV) vector encoding IL-4 into the hippocampus resulted in sustained expression of IL-4, reduced astro/microgliosis, amyloid-beta peptide (Abeta) oligomerization and deposition, and enhanced neurogenesis. Moreover, increased levels of IL-4 improved spatial learning, promoted phosphorylation of N-methyl-D-aspartate receptor subunit 2B at Tyr 1472, and enhanced its cell surface retention both in vivo and in vitro. Our data suggest that neuronal anti-inflammatory cytokine signaling may be a potential alternative target for non-Abeta-mediated treatment of AD.


Assuntos
Doença de Alzheimer/terapia , Precursor de Proteína beta-Amiloide/genética , Sistema Nervoso Central/metabolismo , Interleucina-4/biossíntese , Presenilina-1/genética , Animais , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/farmacologia , Terapia Genética , Hipocampo/metabolismo , Interleucina-4/genética , Interleucina-4/farmacologia , Camundongos , Camundongos Mutantes , Neurônios/química , Neurônios/metabolismo , Transdução de Sinais , Transfecção
15.
FASEB J ; 24(8): 2904-15, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20354135

RESUMO

Tau-tubulin kinase-1 (TTBK1) phosphorylates microtubule-associated protein tau at specific serine/threonine residues found in paired helical filaments (PHFs), and its expression is up-regulated in the brain in Alzheimer disease, suggesting its role in tauopathy pathogenesis. To understand the effects of TTBK1 on tauopathy in vivo, we have developed bigenic mice overexpressing full-length TTBK1 and the P301L tau mutant. The bigenic mice show enhanced tau phosphorylation at multiple sites (AT8, 12E8, PHF-1, and pS422), tauC3-immunoreactive tau fragmentation, and accumulation of tau aggregates in cortical and hippocampal neurons at 12-13 mo of age. However, the phosphorylated tau aggregates were predominantly sarkosyl soluble and migrated in the light sucrose density fraction after discontinuous sucrose gradient ultracentrifugation, which suggests that they form small oligomers. The bigenic mice show significant locomotor dysfunction as determined by both rotorod and grip strength tests, as well as enhanced loss of motor neurons in the L4-L5 spinal cord. This neuronal dysfunction and degeneration was associated with increased levels of tau oligomers, cyclin-dependent protein kinase 5 activators p35 and p25, and pY216 phosphorylated glycogen synthase kinase 3-beta. These data suggest that TTBK1 up-regulation enhances tau phosphorylation and oligomerization, whose toxicity results in enhanced neurodegeneration and locomotor dysfunction in a tauopathy animal model.


Assuntos
Mutação , Degeneração Neural/etiologia , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas tau/metabolismo , Animais , Camundongos , Camundongos Mutantes , Transtornos das Habilidades Motoras/etiologia , Fosforilação , Multimerização Proteica , Tauopatias/etiologia , Regulação para Cima , Proteínas tau/genética
16.
Ageing Res Rev ; 70: 101385, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34098113

RESUMO

Huntington's disease (HD) is an autosomal neurodegenerative disorder caused by extended trinucleotide CAG repetition in the HTT gene. Wild-type huntingtin protein (HTT) is essential, involved in a variety of crucial cellular functions such as vesicle transportation, cell division, transcription regulation, autophagy, and tissue maintenance. The mutant HTT (mHTT) proteins in the body interfere with HTT's normal cellular functions and cause additional detrimental effects. In this review, we discuss multiple approaches targeting DNA and RNA to reduce mHTT expression. These approaches are categorized into non-allele-specific silencing and allele-specific-silencing using Single Nucleotide Polymorphisms (SNPs) and haplogroup analysis. Additionally, this review discusses a potential application of recent CRISPR prime editing technology in targeting HD.


Assuntos
Doença de Huntington , Alelos , Regulação da Expressão Gênica , Marcação de Genes , Humanos , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Doença de Huntington/genética , Doença de Huntington/terapia
17.
Mol Ther ; 17(5): 803-9, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19277012

RESUMO

Accumulation of aggregated amyloid-beta (Abeta) peptide was studied as an initial step for Alzheimer's disease (AD) pathogenesis. Following amyloid plaque formation, reactive microglia and astrocytes accumulate around plaques and cause neuroinflammation. Here brain chemokines play a major role for the glial accumulation. We have previously shown that transgenic overexpression of chemokine CCL2 in the brain results in increased microglial accumulation and diffuse amyloid plaque deposition in a transgenic mouse model of AD expressing Swedish amyloid precursor protein (APP) mutant. Here, we report that adeno-associated virus (AAV) serotype 1 and 2 hybrid efficiently deliver 7ND gene, a dominant-negative CCL2 mutant, in a dose-response manner and express >1,000-fold higher recombinant CCL2 than basal levels after a single administration. AAV1/2 hybrid virus principally infected neurons without neuroinflammation with sustained expression for 6-months. 7ND expressed in APP/presenilin-1 (APP/PS1) bigenic mice reduced astro/microgliosis, beta-amyloidosis, including suppression of both fibrillar and oligomer Abeta accumulation, and improved spatial learning. Our data support the idea that the AAV1/2 system is a useful tool for CNS gene delivery, and suppression of CCL2 may be a therapeutic target for the amelioration of AD-related neuroinflammation.


Assuntos
Precursor de Proteína beta-Amiloide/genética , Amiloidose/terapia , Quimiocina CCL2/genética , Dependovirus/genética , Gliose/terapia , Aprendizagem em Labirinto/fisiologia , Presenilina-1/genética , Peptídeos beta-Amiloides/metabolismo , Animais , Quimiocina CCL2/fisiologia , Feminino , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Transgênicos
18.
Diagnostics (Basel) ; 10(4)2020 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-32316237

RESUMO

This study measures the total graft of 18F-sodium fluoride (NaF) uptake in non-instrumented posterolateral lumbar fusion (niPLF) patients one month after surgery and correlates it with the difference in the clinical findings between the baseline and one year after surgery. The walking distance (WLK-D), visual analog scale of back pain (VAS-B), VAS score of leg pain (VAS-L), tandem test (TAN), Oswestry Disability Index questionnaire (ODI), and European Quality of Life-5 Dimensions questionnaire (EQ-5D) were assessed before surgery and one year after. The graft NaF uptake was analyzed quantitatively with a fixed threshold algorithm resulting in the total graft uptake (SUVtotal) and partial volume corrected SUVtotal (cSUVtotal). Only 4 out of 18 patients experienced fusion; they had an insignificantly lower median total graft uptakes, i.e., 1178 SUVtotal vs. 1224 SUVtotal (p = 0.73) and 1282 cSUVtotal vs. 1231 cSUVtotal (p = 0.35), respectively. Similarly, fused patients experienced insignificantly larger pain decreases, i.e., median VAS-B 4.3 vs. 3.8 (p = 0.92) and VAS-L -6.4 vs. -4.4 (p = 0.2). We found an insignificant trend for a lower NaF uptake and less pain in fused patients. The NaF uptake did not correlate with the chronological change in the clinical parameters.

19.
Spine J ; 20(5): 677-684, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32001384

RESUMO

BACKGROUND CONTEXT: Due to poor bone stock in the elderly, a noninstrumented fusion is commonly performed in Scandinavia when instability is present. Allograft bone is often used as graft extender with consequent low fusion rates. The use of 15 amino acid residue (ABM/P-15) has shown superior fusion rates in dental and cervical spinal surgery but no clinical studies have been conducted in noninstrumented lumbar fusion surgery. PURPOSE: To evaluate patient reported outcomes (PROs) and the intertransverse fusion rate in noninstrumented posterolateral fusion with either ABM/P-15 or allograft. STUDY DESIGN: Double-blind randomized clinical trial. PATIENT SAMPLE: Patients 60 years or older with degenerative spondylolisthesis undergoing decompression and noninstrumented posterolateral fusion. OUTCOME MEASURES: Visual analog scales for back and leg pain, Oswestry Disability Index and EuroQoL-5D. METHODS: One hundred one patients were enrolled in the study and randomized 1:1 to either ABM/P-15 (mixed 50/50, 5cc/level) or allograft bone (30 g/level), both mixed with local bone graft. PROs were collected at baseline and at 12 and 24 months after surgery. The patients underwent 1-year postoperative fine cut computed tomography-scans (0.9 mm) with reconstructions, independently evaluated by three reviewers. Fusion status was concluded by consensus of two of the three as "fusion" or "no fusion." RESULTS: There were 49 patients available for analysis in both cohorts. The two groups were similar in terms of sex distribution, age, and number of levels fused. The fusion rate was significantly higher in the ABM/P-15 group with 50% fused compared with 20% in the allograft group. PROs at baseline and at all follow-up time points were similar between the two groups. CONCLUSIONS: Patients undergoing noninstrumented posterolateral fusion augmented with ABM/P-15 had a statistically significantly higher fusion rate compared with allograft when evaluated with postoperative fine cut computed tomography-scans (0.9 mm) with reconstructions. However, this did not translate to better clinical outcomes.


Assuntos
Fusão Vertebral , Espondilolistese , Aloenxertos , Humanos , Vértebras Lombares/diagnóstico por imagem , Vértebras Lombares/cirurgia , Pessoa de Meia-Idade , Estudos Prospectivos , Países Escandinavos e Nórdicos , Fusão Vertebral/efeitos adversos , Espondilolistese/diagnóstico por imagem , Espondilolistese/cirurgia , Resultado do Tratamento
20.
J Neurosci ; 28(53): 14511-21, 2008 Dec 31.
Artigo em Inglês | MEDLINE | ID: mdl-19118186

RESUMO

Tau-tubulin kinase-1 (TTBK1) is involved in phosphorylation of tau protein at specific Serine/Threonine residues found in paired helical filaments, suggesting its role in tauopathy pathogenesis. We found that TTBK1 levels were upregulated in brains of human Alzheimer' disease (AD) patients compared with age-matched non-AD controls. To understand the effects of TTBK1 activation in vivo, we developed transgenic mice harboring human full-length TTBK1 genomic DNA (TTBK1-Tg). Transgenic TTBK1 is highly expressed in subiculum and cortical pyramidal layers, and induces phosphorylated neurofilament aggregation. TTBK1-Tg mice show significant age-dependent memory impairment as determined by radial arm water maze test, which is associated with enhancement of tau and neurofilament phosphorylation, increased levels of p25 and p35, both activators of cyclin-dependent protein kinase 5 (CDK5), enhanced calpain I activity, and reduced levels of hippocampal NMDA receptor types 2B (NR2B) and D. Enhanced CDK5/p35 complex formation is strongly correlated with dissociation of F-actin from p35, suggesting the inhibitory mechanism of CDK5/p35 complex formation by F-actin. Expression of recombinant TTBK1 in primary mouse cortical neurons significantly downregulated NR2B in a CDK5- and calpain-dependent manner. These data suggest that TTBK1 in AD brain may be one of the underlying mechanisms inducing CDK5 and calpain activation, NR2B downregulation, and subsequent memory dysfunction.


Assuntos
Regulação para Baixo/genética , Deficiências da Aprendizagem/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas Serina-Treonina Quinases/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Actinas/metabolismo , Fatores Etários , Doença de Alzheimer/patologia , Animais , Calpaína/metabolismo , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/patologia , Hipocampo/metabolismo , Humanos , Espectrometria de Massas , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Peso Molecular , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Comportamento Espacial/fisiologia , Transfecção , Regulação para Cima , Região do Complexo-t do Genoma
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